The Ectoparasitic Honey Bee Mite Varroa Jacobsoni, First Described By

A COMPARATIVE ANALYSIS OF SHAKING SOLUTIONS FOR THE DETECTION OF VARROA JACOBSONI ON ADULT HONEYBEES (1) D. DE JONG D. DE ANDREA ROMA L. S. GONÇALVES ( SUMMARY Various liquids could be used for the detection of Varroa jacobsoni on honey bees using the shaking method. Mechanical shaking for 30 minutes in a rotary shaker disloged all but 3 mites in 27 separate examinations. The ectoparasitic honey bee mite Varroa jacobsoni, first described by OUDEMANS (1904) from the Asian honey bee Apis cerana in Java, is distributed throughout Asia, wherever A. Cerana occurs (DE JONG and MORSE 1979). Since the 1950’s, and probably earlier, beekeepers have moved colonies of European bees, A. mellifera, into Asia (CRANE 1978) and in some cases colonies of A. cerana (infested with Varroa) from Asia into Europe (LANGHE and NATSKII 1976, RUTTNER and RITTER, 1980) and, as a result of contact between the two bee species, V. jacobsoni has moved to A. mellifera as a new host (CRANE 1978, MORSE, 1978). Although A. cerana is not seriously affected by the mites, colonies of A. mellifera are severely compromised (ALPATOV 1977). Within the last twenty years Y. jacobsoni has extended its range from Asia to Eastern and Western Europe. Northern Africa and much of South America (CRANE 1978, 1979). The mites cause damage to adult and developing honey bees as they feed on the bee’s hemolymph. In colonies with low populations, the developing bees may lose 5 to 25 % of their weight; occasionally there is damage to wings or other appendages. However, the signs are easily missed as only a small percentage of emerging bees are obviously deformed even when over 50 % of the brood cells are infested (DE JONG et (I) This investigation was supported by N.S.F. Grant No. D.A.R. - 7920922 to R. A. MORSE and grants from the Foundation for the Support of Research in the State of Sao Paulo (F.A.P.E.S.P.) and the Brazilian Research Council (C.N.P.Q.) to L. S. GONCALVES. (2) Department of Entomology, Cornell University, Ithaca, NY 14853, U.S.A. (3) Depto. de Genetica, Faculdade de Medicina, U.S.P., 14.100 RibeirAo Preto. SP Brazil. (4) Faculdade de Filosophia, Ci6ncias e Letras, U.S.P., 14.100 Ribeir5o Preto, SP Brazil. al., submitted for publication). Most of the damaged bees are immediately removed from the colony by house-cleaning bees (DE JONG, unpublished data). When infestations have increased to the point that they are noticeable, the mite populations are already quite high (DE JONG and GONÇALVES 1981). During the three to six years necessary for a new infestation to reach this point, the mites are widely dispersed by drifting bees, swarms and the movement of colonies by man (PHADxE et al., 1966, SMIRNOV 1978). It is therefore desirable to locate new foci of infestation early so that the mites can be controlled or eliminated. There are two principal ways of examining bee colonies for the presence of Y. jacobsoni. One method is to uncap brood cells and remove the larvae or pupae for examination. Since the mites have a preference for drone brood (RITTER and RUTTNER 1980), examining drone cells is usually more productive. A second method involves an examination of the adult bees by shaking them in a solution which will dislodge the mites. Large numbers of bees can be sampled in a short time by this method, facilitating the detection of low-level infestations and providing a means for estimation of mite populations. Varroa jacobsoni that are attached superficially to the bee are easy to remove by shaking bees in a liquid phase. However, mites that are attached to the intersegmental membranes of the abdominal sternites or tergites are difficult to see and to remove even if the bees are dead. Several types of solutions have been used for shaking bees to detect V. jacobsoni. These include hot water (GROBOV 1977), detergent solutions (STOLBOV and VASIxov 1976), hexane or gasoline (RITTER and RUTTNER 1980) and 96 % ethanol (DE JONG and GONÇALVES 1981). Gasoline is reportedly nearly 100 % effective for separating mites from adult bees (RITIER and RUTTNER 1980); however, it is dangerous to use as the fumes are toxic and highly flammable. In Brazil ethanol was found equally effective (GONCALVEs et al., 1980), but in most other countries it may be costly and not readily available. Since the shaking technique is used frequently for the detection of V. jacobsoni, we decided to test the various products to compare their efficiency for extracting mites. Solutions were selected according to availability and facility of use. We tested each solution with and without the use of a laboratory shaker. Dried samples were also tested to determine the suitability of winter-killed bees or dried samples sent to a central laboratory for diagnosis. MATERIALS AND METHOD A plastic shaking container developed in Brazil (DE JONG and GONÇALVES 1981) was used for all of the experiments. It consisted of a 1-1 plastic alcohol bottle that had a constriction in the middle. The middle third section of the coastruction was cut out and the portions reassembled. A round piece of wire screening with openings 3 mm square (these should be larger than 1.9, but smaller than 4.5 mm) was cut slightly larger than the inside diameter of the bottle, then the screen was fitted into the upper end of the bottle from the inside. With the bottle cap in place the inverted bottle assembly retained the bees above the screen while the mites dropped down to the neck of the bottle (Fig. 1). Where such a container is not available, a shaking container can be made by cutting the bottom off any plastic bottle and using a piece of flexible plastic wrap secured with a piece of elastic as a cover for the bottom. Several small holes about 0.5 mm diameter should be made in the top of the shaker or in the plastic wrap to allow for the release of air pressure caused by the evaporation of fluids during the shaking process. Adult bee samples were collected by brushing them from brood combs into a beaker or other wide-mouth container with 150 ml of shaking solution. The bees and solution were then poured into the shaking container (with cap in place) and enough extra solution added to cover all of the bees. Each bottle was agitated by hand with the cap down and moved in a circular motion for one minute. The solution was then poured into a bowl lined with a white small mesh cloth. The cloth was then lifted from the bowl and the mites on the cloth counted. The bottle was then recapped and refilled with the same solution and subjected to 30 minutes of circular agitation in a laboratory shaker. Additional mites separated by the second shaking were counted as before. As a final check all of the bees were examined individually for remaining mites. In a separate test, two samples of live bees were placed into empty jars and killed by freezing overnight. One sample was dried in the sun for 24-hr. at 24-27 °C and the other oven-dried at 60 °C for 2-hr. The bees were then subjected to the shaking procedure using 96 % ethanol. RESULTS AND DISCUSSION The mean number of mites per samples was .156 per bee, with a range of .0237 to 583 (Table 1). Hand-shaking for one minute was reasonably effective, but for all solutions tested, mechanical shaking for 30 minutes effectively removed 100 % of the mites. Only rarely were mites encountered on bees after they had been through the mechanical shaking. Several of the solutions were inconvenient to use. Many of the bees in water 25 °C (77 °F) were able to crawl out of the container. This was less of a problem with water at 40 °C (104 °F). The hot water, 60 °C (140 °F), and 100 °C (212 °F) was difficult to handle without an insulated container. Bees quickly succumbed in the detergent solutions as well as in the ethanol and isopropyl alcohol. The foam in the detergent solutions sometimes made it difficult to count mites, though rinsing the filter cloth with additional water usually eliminated this problem. The results from the various solutions were compared to those obtained with 96 % ethanol to determine if they were equally effective in extracting the mites using only hand-shaking. Water, Tween-80, and 15 % ethanol were significantly less efficient. However, there was no significant advantage to using 96 % ethanol over the remaining alcohol solutions. The shaking procedure using dried bee samples proved unreliable. Over 10 °/> of the mites remained attached to the dried bees even after 30 minutes of mechanical shaking in 96 % ethanol (Table 2). In contrast, 100 % of the mites were dislodged from freshly killed bees by the same treatment. CONCLUSIONS Shaking bees in various solutions is an efficient means of detecting the presence of V. jacobsoni in samples of freshly killed adult honey bees. Twenty-five percent solutions of ethanol or isopropyl alcohol were effective, convenient to use, and reasonable in cost. Hand-shaking for one minute removed 79-96 % (average 92 %) of the mites when 25 % alcoholic solutions were used. Mechanical shaking on a rotary shaker for 30 minutes using 25 % alcoholic solutions removed 100 °/> of the mites. The difference in efficiency of mite removal between hand-shaking and mechanical shaking was statistically significant. Dried adult bee samples, which are more easily shipped to a central laboratory, can be diagnosed with a shaking solution, although the method is not as reliable as when freshly killed bees are used.

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